A group of biological agents termed superantigens has been described based on their ability to stimulate monocytes and unprimed CD4+ (MHC Class II restricted) and CD8+ (MHC Class I restricted) T-cells. Included with the designation of superantigens are the enterotoxins of Staphylococcus aureus. 
The enterotoxins of Staphylococcus aureus form a group of serologically distinct proteins, originally designated A, B, C1, C2, C3, D, E, G, and H. Subsequently, a number of variants have been described. These proteins and toxic shock syndrome proteins were originally recognized as the causative agents of staphylococcal food poisoning. Ingestion of preformed enterotoxin in contaminated food leads to the rapid development (within two to six hours) of symptoms of vomiting and diarrhea that are characteristic of staphylococcal food poisoning. Toxic shock syndrome toxin-1, TSST- 1, a distantly related protein also produced by S. aureus, is classically responsible for the toxic shock syndrome, although other staphylococcal enterotoxins may result in the syndrome due to the induction of cytokines.
Other agents which have been identified as superantigens include for example, the staphylococcal exfoliative toxins, A and B; the mammary tumor virus superantigen; rabies virus nucleocapsid protein; pyrogenic exotoxins A, B, C from S. pyrogens; and the Mycoplasma arthritides mitogen. Additional biological agents which have been demonstrated to have superantigen properties include the human immunodeficiency virus (HIV), gp120 and peptides from HIV, mouse mammary tumor virus and feline immunodeficiency virus. A Leishmania peptide antigen has also been disclosed as a superantigen.
Superantigens, unlike conventional antigens, do not require processing in-vivo. In general, superantigens have two binding regions, one of which interacts with the Class II major histocompatibility complex (MHC) on the antigen presenting cell and the other which interacts with the Vβ variable region of the T-cell receptor on CD4 and/or CD8 cells. Various enterotoxins bind to one or more of the different Vβ receptor epitopes. In contrast to conventional antigens, superantigens do not occupy the T-cells receptor cleft but are felt to bind to an external region thus explaining the ability to activate a broad population of T-cells.
Enterotoxins produced by Staphylococcus aureus include a group of related proteins of about 20 to 30 Kd. The complete amino acid composition of a number of staphylococcal enterotoxins and streptococcal pyrogenic exotoxin has been reported (see e.g., PCT Patent Appl. No. WO 93/24136.)
Staphylococcal enterotoxins (“SEs”) were initially classified on the basis of their antigenic properties into groups A, B, C1, C2, C3, D, and E. Subsequent relatedness was based on peptide and DNA sequence data. Among the staphylococcal enterotoxins, groups B and C are closely related and groups A, D, and E are closely related in amino acid sequence. SEC1, SEC2, and SEC3 and related isolates share approximately 95% sequence similarity. Table 1 shows the alignment of the predicted sequences of the eight known SEC variants following cleavage of the signal peptide. The N-terminus of each of the mature proteins was verified by amino acid sequencing. Amino acid positions that contain residues that are not conserved among all SEC are indicated by asterisks. SEB and SEC are approximately 45-50% homologous. In contrast, non-enterotoxin superantigens, TSST-1 and Streptococcal Pyrogenic Enterotoxin C (SPEC) share only approximately 20% primary sequence homology to SEC. Despite these differences, the tertiary structure of the various enterotoxins show nearly identical folds.
The staphylococcal enterotoxins A, B, C1, C2, C3, D, E, G and H share a common structural feature of a disulfide bond not present in other enterotoxins. Table 2 shows the position of the disulfide bond in a number of enterotoxins. Data in reference to the active sites of the enterotoxin molecule in relationship to biological activity, MHC binding, and TCR binding has been obtained. Sequence data demonstrate a high degree of similarity in four regions of the enterotoxins (See Table 3). The peptides implicated in potential receptor binding correspond to regions 1 and 3 which form a groove in the molecule. Amino acid residues within and adjacent to the α3 cavity of SEC3 have been shown to relate to T-cell activation.
TABLE 2LOCATION OF DISULFIDE LOOP IN STAPHYLOCOCCUS ENTEROTOXINSAMINOACIDENTEROTOXINRESIDUESAMINO ACID SEQUENCE OF DISULFIDE LOOPSEA96-10696?CAGGTPNKTAC(SEQ. ID. NO:9)SEB93-11493?CYFSKKTNDINSHQTPKRKTC(SEQ. ID. NO:10)SEC193-11093?CYFSSKDNVGKVTGGKTC(SEQ. ID. NO:11)SEC293-11093?CYFSSKDNVGKVTGGKTC(SEQ. ID. NO:12)SEC3 FRI 91393-11093?CYFSSKDNVGKVTGGKTC(SEQ. ID. NO:13)SEC3 FRI 90993-11093?CYFSSKDNVGKVTSGKTC(SEQ. ID. NO:14)SEC 444693-11093?CYFSSKDNVGKVTGGKTC(SEQ. ID. NO:15)SEC-Bovine93-11093?CYFSSKDNVGKVTGGKTC(SEQ. ID. NO:16)SEC-Ovine93-11093?CCFSSKDNVGKVTGGKTC(SEQ. ID. NO:17)
The staphylococcal enterotoxins are potent activators of T-cells, resulting in proliferation and the generation of cytotoxic T-cells. SEA is a potent T-cell mitogen eliciting strong polyclonal activation at concentrations of 10−13 to 10−10 molar in human systems.
The staphylococcal enterotoxins, aside from the acute gastroenteritis and toxic shock syndrome associated with them, have been shown to have a variety of other beneficial biological effects. The biological effects of these agents and the toxic shock syndrome are due in part to the ability of staphylococcal enterotoxins to induce cytokines. Various cytokines described include IL-1, IL-2, and tumor necrosis factor (“TNF”). More recently SEB and toxic shock syndrome toxin (“TSST-1”) have been shown to induce interleukin-12, an inducer of cell mediated immunity, in human peripheral blood mononuclear cells. (See Leung et al., J Exp Med, 181:747 (1995)). The antitumor activity of treating cancer in rabbits utilizing 40 to 60 μg/kg of a staphylococcal enterotoxin has been disclosed in PCT Patent Appl. Nos. WO 91/10680 and WO 93/24136.
Exposure to enterotoxin either in-vitro or in-vivo leads to depletion of T-cells having the appropriate Vβ receptor through programmed cell death in some strains of mice, specifically Balb/c and CBA/2. Cell death can be prevented by high doses of retinol or RU-38486. Programmed cell death has not been observed upon exposure of human cells to enterotoxins.
Although the systemic lethal toxicity of enterotoxins has been related to their ability to induce cytokines, particularly IL-1, IL-2 and gamma interferon, lethal toxicity also appears to be related to a synergistic activity with endogenous endotoxins and the ability of the liver to detoxify endotoxins. Although a number of animals have been utilized to evaluate lethality, the accepted model is the continous infusion over a period of time, usually 4 days, in rabbits. The direct toxic dose varies among various species. The 50% lethal dose of TSST-1 is approximately 50 μg/kg for Balb/c mice. Piglets, although showing clinical manifestations of toxic shock syndrome, tolerate doses of 100 μg/kg of TSST-1. TSST-ovine is known to be non-toxic at doses of 200 μg in rabbits.
In Dutch belted rabbits, intramuscular injection of 50 mg/kg of staphylococcal enterotoxin B caused death. Intravenous injection at 0.5 to 1.0 mg/kg of enterotoxin A or B in rhesus macaques results in hypotension and death (Liu C. T., et al Amer J Vet Res 39:279 and 1213, 1978).
In contrast to other species, man is extremely sensitive to enterotoxins. One (1) mg of TSST-1, approximately 15 nanogram/kg, can be lethal for man. Therefore, the recommended doses currently proposed in the art for treating man are unacceptable. There is a need, therefore, for mutant staphylococcal enterotoxins which are non-toxic at anticipated doses for man while still retaining desirable biological activity.
Several studies of staphylococcal enterotoxin have identified a number of biologically active modified or mutant enterotoxins with reduced toxicity. Carboxymethylation of SEB results in a loss of gastrointestinal toxicity but not mitogenic activity. Studies with the TSST-1 have demonstrated the active site to be between amino acids residue 115 and 141. Point mutation of site 135 from histidine to alanine results in a loss of mitogenic activity and toxicity (See Bonventre P. F., et al. Infect Immun 63:509(1995)). Studies with the staphylococcal enterotoxin SEC1 demonstrated that the disulfide bond between residue 93 and 110 is not required for activity (See Hovde et al., Mol Microbiol 13:897 (1994)). Studies of the molecular binding region of staphylococcal enterotoxin B using overlapping peptides demonstrated peptide 124 to 154 inhibited SEB induced mitogenic activity.
Based on the known biological activities of the toxic native enterotoxins, it is desirable to create mutants which are at least 1000-fold or more less toxic compared to native enterotoxins and retain biological activity. Recent studies have demonstrated that mutant enterotoxins can be produced which retain certain biological activities and which may be significantly less lethal as determined in rabbits. A mutant of the TSST-1 enterotoxin which differs in amino acid 136 and is non-lethal at ten times the lethal dose of the native toxin (in rabbits), but retains biological activity has been disclosed. A number of mutants of SEC1, unable to form a disulfide bond, have been reported to be ten times less toxic than the native toxin while retaining biological activity. (See e.g., Hovde et al., Molec Microbiol 13:897 (1994)).